We are interested in exploring the interactions between the immune system of the human malaria vector Anopheles gambiae and Plasmodium parasites to establish how they affect vector competence. Some major areas of interest include:[unreadable] [unreadable] Reactive oxygen species (ROS) and the balance between fecundity and immunity in An. gambiae:[unreadable] The effect of aging on reproductive output in An. gambiae females from three strains that differ in their ability to melanize Plasmodium and in their systemic levels of hydrogen peroxide (H2O2) was analyzed. The number of mosquito oocytes that develop after a blood meal decreases with age in all mosquito strains. This decline is more pronounced in unselected (G3) females and in a strain selected to be refractory (R) to Plasmodium than in a highly susceptible (S) strain. Reduction of ROS levels in G3 and R females by administration of anitoxidants restores this age-related decline in fecundity. The S and G3 strains are fixed for two catalase alleles that differ in their specific activity and Km, probably due to differences in stability of the active tetrameric form of the enzyme. A systemic reduction in catalase activity by double-stranded RNA (dsRNA)-mediated knockdown significantly reduced the reproductive output of mosquito females, indicating that catalase plays a central role in protecting the oocyte and early embryo from ROS damage. This work as published in PNAS.[unreadable] [unreadable] Strains of An. gambiae with higher systemic levels of ROS survive better a bacterial challenge, while reduction of ROS by dietary administration of antioxidants significantly decreases survival, indicating that ROS are required to mount effective antibacterial responses. Expression of several ROS detoxification enzymes increases in the midgut and fat body following a blood meal, and the inductions are significantly higher after P. berghei infection. Paradoxically, a complete lack of induction of catalase mRNA and lower catalase activity was observed in P. berghei-infected midguts. This suppression of midgut catalase expression is a specific response to ookinete midgut invasion and is expected to lead to higher local levels of hydrogen peroxide. Further reduction of catalase expression by dsRNA-mediated gene silencing promotes parasite clearance by a lytic mechanism. We conclude that ROS modulate An. gambiae immunity, and that the mosquito response to P. berghei involves a local reduction of hydrogen peroxide detoxification in the midgut that contributes to limit Plasmodium infection. The manuscript describing this work has been submitted to the Journal of Biological Chemistry.[unreadable] [unreadable] Mosquito Midgut Epithelial Cells responses to Plasmodium Invasion: [unreadable] Previous studies from our group indicate that inducible peroxidases mediate nitration of mosquito midgut cells undergoing apoptosis in response to P. berghei invasion. We hypothesize that the higher steady-state levels of H2O2, a substrate required by heme-peroxidases, present in the refractory (R) strain accelerates peroxidase-mediated reactions and cause parasite damage. We found that, indeed, apoptotic responses and tissue repair are accelerated in R relative to G3 females. At 30-32h post-feeding invaded cells are undergoing nitration and apoptosis in G3, while in R females damaged cells have already budded-off into the midgut lumen and very little evidence of tissue damage is left. The extent to which accelerated nitration contributes to ookinete damage is under investigation.[unreadable] [unreadable] We have characterized a heme-peroxidase that is produced by midgut epithelial cells and has an immunomodulatory role, we refer to it as immunomodulatory peroxidase 1 (Imper1). Imper1 mRNA expression increases dramatically following a blood-meal and the protein product is secreted into the midgut lumen. Imper1 silencing results in activation of the Rel2 pathway in midgut cells, resulting in a strong anti-bacterial response that prevents the normal proliferation of the midgut bacterial flora. Silencing of Imper1 also results induces nitric oxide synthase (NOS) expression in the midgut and dramatically reduces P. berghei and P. falciparum infection. This effect is independent of bacteria, as it is also observed in antibiotic-treated mosquitoes in which antibacterial responses are no longer activated. Furthermore, double silencing of Imper1 and NOS rescues parasite survival to control levels.[unreadable] [unreadable] A Anopheles culicifacies line that is refractory (R) to Plasmodium vivax infection has been identified in India. An immune-responsive serine protease (IRSP) gene is expressed at very high levels in R when compared to a susceptible (S) strain. We cloned and silenced the orthologue of IRSP in An. gambiae to test the hypothesis that this gene mediates refractoriness to Plasmodium in this mosquito species. Unexpectedly, silencing of IRSP significantly decreases P. berghei and P. falciparum infection. The phenotypes observed in all strains tested are opposite to those reported when a serine protease inhibitor (SRPN6) is silenced, suggesting that IRSP and SRPN6 may have antagonistic functions. The interaction of IRSP with this and other serpins is under investigation.[unreadable] [unreadable] [unreadable] STAT signaling pathways and immune responses to Plasmodium: [unreadable] We have identified two transcription factors from the signal transduction and activation of transcription (STAT) family in A. gambiae, AgSTAT-A and AgSTAT-B; as well as orthologues of two suppressors of these pathways in vertebrates, SOCS and PIAS. All these genes are expressed in the immune-responsive A. gambiae Sua 5.1 cell line and in adult females, and NOS and SOCS are transcriptionally activated in response to bacterial challenge. Silencing of either AgSTAT-A or AgSTAT-B in the cell line or in vivo decreases NOS and SOCS mRNA expression after bacterial challenge. AgSTAT-A regulates basal levels of AgSTAT-B expression, as silencing of AgSTAT-A also reduces AgSTAT-B mRNA levels. In vivo silencing of AgSTAT-B by dsRNA injection significantly increases P. berghei and P. falciparum infection and decreased NOS, Tep1 and SOCS mRNA expression. Furthermore, silencing of SOCS has the opposite effect and dramatically reduces infection. The differences in expression in infected mosquitoes in which the AgSTAT-B is silenced will be evaluated using microarray analysis to identify new effectors of the AgSTAT-B pathway. A manuscript describing this work is in preparation.[unreadable] [unreadable] Identification of mosquito genes required for Plasmodium oocyst development: [unreadable] In a collaborative project with Dr. David Schneider, we have cloned and silenced five A. gambiae orthologues (and one homologue) of six Drosophila genes identified in a genetic screen, that are required for injected Plasmodium gallinaceum ookinetes to develop into mature oocysts in the fly. For five genes silencing affects Plasmodium berghei development in A. gambiae, but in two cases, the effect is the opposite from that in Drosophila, and gene silencing enhanced infection. Surprisingly, for three genes silencing had a similar effects on P. berghei and P. falciparum infection A. gambiae, but in three other cases it the opposite effect in these two parasite species. We conlcude that the responses observed after P. berghei infection may often not predict those to P. falciparum. We are further investigating two genes that limit P. falciparum infection, a tetraspanin and a glutathione S transferase, as well as a gene involved in oxidation stress resistance, which mediates the induction of ROS detoxification enzymes in response to blood feeding.